User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/04/15: Difference between revisions

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==Entry title==
=='''SDS Page Gel Electrophoresis'''==
* Insert content here...


#4 - 12 well pre-cast Mini Protean TGX gel was obtained and prepared.
**The comb and tape was removed from each gel
**The wells of each gel were rinsed with the prepared running buffer dilution
#The Bio-Rad Mini Protean system electrophoresis cell was assembled.
** The inner and outer buffer chambers were filled with the diluted running buffer to the "4 gel" mark.
#Each of the 4 gels were loaded with each of the 20 μL reaction samples prepared yesterday in the following manner:
**The samples were heated for 5 min at 100C in the thermocycler pre-loading
'''Gel AO'''
==<font color=#0f1028></font>==
{|style="width:700px"
|<u>'''Well'''</u>
|<u>'''Sample'''</u>
|-
|1
|Ladder
|-
|2
|BLANK
|-
|3
|1 μM Proteinase K (I)
|-
|4
|100 nM Proteinase K (I)
|-
|5
|10 nM Proteinase K (I)
|-
|6
|1 nM Proteinase K (I)
|-
|}
'''Gel AI'''
==<font color=#0f1028></font>==
{|style="width:700px"
|<u>'''Well'''</u>
|<u>'''Sample'''</u>
|-
|1
|Ladder
|-
|2
|BLANK
|-
|3
|1 μM Proteinase K (II)
|-
|4
|BLANK
|-
|5
|100 nM Proteinase K (II)
|-
|6
|BLANK
|-
|7
|10 nM Proteinase K (II)
|-
|8
|BLANK
|-
|9
|1 nM Proteinase K (II)
|-
|}
'''Gel BI'''
==<font color=#0f1028></font>==
{|style="width:700px"
|<u>'''Well'''</u>
|<u>'''Sample'''</u>
|-
|1
|Ladder
|-
|2
|BLANK
|-
|3
|1 μM Trypsin
|-
|4
|100 nM Trypsin
|-
|5
|10 nM Trypsin
|-
|6
|1 nM Trypsin
|-
|7
|BLANK
|-
|8
|1 μM Chymotrypsin
|-
|9
|100 nM Chymotrypsin
|-
|10
|10 nM Chymotrypsin
|-
|11
|1 nM Chymotrypsin
|-
|}
'''Gel BO'''
==<font color=#0f1028></font>==
{|style="width:700px"
|<u>'''Well'''</u>
|<u>'''Sample'''</u>
|-
|1
|Ladder
|-
|2
|BLANK
|-
|3
|1 μM Thermolysin
|-
|4
|SKIP
|-
|5
|100 nM Thermolysin
|-
|6
|SKIP
|-
|7
|10 nM Thermolysin
|-
|8
|BLANK
|-
|9
|1 nM Thermolysin
|-
|}
IV. The gel was run for approximately 10 min at 200 V, 0.05 A, and 10 W.
'''NOTE:''' The top of the chamber was not functioning properly in that a closed circuit was not established and the top had to be manually held/pushed down for the entirety of the run.
V. After 10 min, the gel was placed in a Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes
VI. The gel was then placed in a Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for approximately 24 hours
VII.Finally, the gel was placed in Destain Solution (10% acetic acid, 90% water) three times for 15 minutes each and the following results were obtained:
[[Image:11134235_10205059533466235_680314354_n_%281%29.jpg|500px]]
[[Image:11149169_10205059536186303_2003984602_n.jpg|500px]]
[[Image:11123991_10205059535426284_1373617103_n.jpg|500px]]


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Revision as of 13:09, 18 April 2015

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SDS Page Gel Electrophoresis

  1. 4 - 12 well pre-cast Mini Protean TGX gel was obtained and prepared.
    • The comb and tape was removed from each gel
    • The wells of each gel were rinsed with the prepared running buffer dilution
  1. The Bio-Rad Mini Protean system electrophoresis cell was assembled.
    • The inner and outer buffer chambers were filled with the diluted running buffer to the "4 gel" mark.
  1. Each of the 4 gels were loaded with each of the 20 μL reaction samples prepared yesterday in the following manner:
    • The samples were heated for 5 min at 100C in the thermocycler pre-loading


Gel AO

Well Sample
1 Ladder
2 BLANK
3 1 μM Proteinase K (I)
4 100 nM Proteinase K (I)
5 10 nM Proteinase K (I)
6 1 nM Proteinase K (I)


Gel AI

Well Sample
1 Ladder
2 BLANK
3 1 μM Proteinase K (II)
4 BLANK
5 100 nM Proteinase K (II)
6 BLANK
7 10 nM Proteinase K (II)
8 BLANK
9 1 nM Proteinase K (II)


Gel BI

Well Sample
1 Ladder
2 BLANK
3 1 μM Trypsin
4 100 nM Trypsin
5 10 nM Trypsin
6 1 nM Trypsin
7 BLANK
8 1 μM Chymotrypsin
9 100 nM Chymotrypsin
10 10 nM Chymotrypsin
11 1 nM Chymotrypsin


Gel BO

Well Sample
1 Ladder
2 BLANK
3 1 μM Thermolysin
4 SKIP
5 100 nM Thermolysin
6 SKIP
7 10 nM Thermolysin
8 BLANK
9 1 nM Thermolysin

IV. The gel was run for approximately 10 min at 200 V, 0.05 A, and 10 W.

NOTE: The top of the chamber was not functioning properly in that a closed circuit was not established and the top had to be manually held/pushed down for the entirety of the run.

V. After 10 min, the gel was placed in a Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes

VI. The gel was then placed in a Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for approximately 24 hours

VII.Finally, the gel was placed in Destain Solution (10% acetic acid, 90% water) three times for 15 minutes each and the following results were obtained:



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